Signaling through the epidermal growth factor receptor (EGFR) has critical roles in development and in diseases such as cancer. All ligands of the EGFR are made as membrane-anchored precursors whose ectodomain must be proteolytically released or shed to trigger EGFR-signaling. EGFR-ligand shedding is therefore crucial for EGFR-signaling. The membrane-anchored metalloproteinase ADAM17 has emerged as the principal regulator of the bioavailability of EGFR-ligands. Mice lacking ADAM17 phenotypically resemble those lacking the EGFR, providing genetic evidence for the essential role of ADAM17 in EGFR signaling and establishing ADAM17 as an important potential target for the treatment of EGFR-dependent cancers. ADAM17 activity is highly regulated and is influenced by numerous signaling pathways. How these pathways functionally intersect with ADAM17 and how ADAM17 is activated are key questions that are the subject of this application. Previously, we had found that ADAM17 is regulated by its transmembrane domain (TMD), but the underlying mechanism remained enigmatic. In a major breakthrough in the last funding period, we identified the seven-membrane spanning iRhoms1 and 2 (iR1, iR2) as novel regulators of ADAM17. We showed that iR2 controls the maturation and function of ADAM17 in immune cells, and that inactivation of iR1 and iR2 abolishes all functions of ADAM17 in mice, thereby providing biochemical, cell biological and genetic evidence that iR1 and iR2 are the long sought-after regulators of ADAM17. The main goals of the current proposal are to understand how iR1 and 2 integrate and interpret the signals that drive the activation of the ADAM17/EGFR pathway and to understand the functional consequences of these interactions in the context of ADAM17-dependent pathological neovascularization. We propose the following specific aims: Aim 1 is focused on understanding how iR1 and iR2 interact with and regulate ADAM17, with an emphasis on the TMD and juxtamembrane domains of both interacting partners. In addition, we will characterize the intracellular maturation and subcellular localization of the iR2/ADAM17 complex. The main goal of aim 2 is to understand how the C-terminal TMDs of iR1 and 2 control their substrate selectivity and to identify what makes a substrate such as HB-EGF selective for iR2. Moreover, we will search for cytoplasmic proteins that interact with and regulate iR2/ADAM17 but cannot bind to the iR2-cub cytoplasmic deletion mutant that inactivates iR2/ADAM17, and will combine these efforts with an analysis of iR1-/-iR2-cub mice. Finally, aim 3 will rigorously test the role of the iR1/2/ADAM17 signaling axis in the context of pathological angiogenesis in mouse models for cancer and proliferative eye diseases. Together, these aims will resolve the most pressing current questions regarding the regulation of ADAM17, a major cellular sheddase that is a critical molecule in EGFR signaling and a target for treatment of cancer, by the newly discovered upstream regulators iR1 and iR2.